1. Field of the Invention
The present invention relates to a transgenic plant with glycosyltransferase gene introduced therein, and the use thereof. More specifically, the invention relates to a technique for synthetically preparing a lipid or a protein with a sugar chain modification intrinsic to animals at a state completely free of any risk of animal-derived infections, in a biological organism such as plant. In accordance with the invention, it is provided a technique for extending the biological sugar chain of a lipid or a protein in animal-, plant- and microbial cells via the introduction of glycosyltransferase gene.
2. Description of the Related Art
Sphingoglycolipids of animal type are utilized as chemical agents for raw materials of pharmaceutical products and cosmetics. Additionally because the sugar chain thereof works as a receptor for various viruses such as influenza virus and bacteria toxins (Karlson, K. A. Animal glycosphingolipids as membrane attachment site for bacteria, Ann. Rev. Biochem., 58, 309-350, 1989), practical application of sphingoglycolipids as novel therapeutic agents with no content of antibiotics or synthetic drug substances has been desired against these infections at an early stage (Tokuhyo 2003-535965, Tokuhyo Hei 10-50347). It is known for example that ceramide trihexoside is a receptor for vero toxin (the toxin generated by Escherichia coli O-157) and Shiga Toxin (the toxin generated by Shigella) for their binding onto cell surface (Cohen, A., Hannigan G E, Williams B R, Lingwood C A, J. Biol. Chem., Dec. 15, 1987; 262(35): 17088-91. Related Articles, Links Roles of globotriosyl- and galabiosylceramide in vero toxin binding and high affinity interferon receptor; Lindberg, A A, Brown J E, Stromberg N, Westling-Ryd M, Schultz J E, Karlsson K A, J. Biol. Chem., Feb. 5, 1987; 262(4): 1779-85. Related Articles, Links Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type I.)
The research works of Watarai, et al. (Watarai, S. et al. Inhibition of vero cell cytotoxic activity in Escherichia coli 0157: H7 lysates by globotriaosylceramide, Gb3, from bovine milk. Biosci. Biotechnol. Biochem., 65, 414-419 (2001)) indicate that bovine-derived ceramide trihexoside added to a culture cell traps vero toxin to inhibit the binding of the toxin to the cell so that vero toxin is inactivated. This indicates a possibility of the pharmaceutical application of ceramide trihexoside. Ceramide trihexoside is abundant in animal tissues (in brain, in particular). However, the related-art method of the extraction thereof from animal tissues is problematic because of infections such as BSE (bovine spongiform encephalopathy). This can be prepared synthetically by an artificial method. However, the method is tough and highly costly. Thus, the method is disadvantageously inappropriate for the mass-scale production thereof (Hasegawa, A., Morita, M., Kojima, Y., Ishida, H. & Kiso, M., Synthesis of cerebroside, lactosylceramide, and ganglioside GM3 analogs containing β-thioglycosidically linked ceramide. Carbonhydr. Res., 214, 43-53 (1991)).
Various reports tell about research examples of the production of useful substances using genetic recombinant plants (Voelker, T. A. et al.: Fatty acid biosynthesis redirected to medium chains in transgenic oil seed plants, Science, 257, 72-74 (1992); Sayanova, O. et al.: Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Δ6-desaturated fatty acids in transgenic tobacco, Proc. Natl. Acad. Sci. U.S.A., 94, 4211-4216 (1997); Ye, X. et al.: Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm, Science, 287, 303-305 (2000); Datta, K.: Bioengineered ‘golden’ indica rice cultivars with β-carotene methabolism in the endosperm with hygromycin and mannose selection systems, Plant Biotech. J., 1, 81-90 (2003); Tozawa, Y. et al.: Characterization of rice anthranilate synthase α-subunit genes OASA 1 and OASA2. Tryptophan accumulation in transgenic rice expressing a feedback-insensitive mutant of OASA1, Plant Physiology, 126, 1493-1506 (2001)). Such types of methods are advantageously low in cost with no generation of carbon dioxide, causing no concerns of any risk of animal infections.
Ceramide trihexoside is one of biosynthetic products of sphingoglycolipids with various physiological activities. It is shown that ceramide trihexoside can synthetically be prepared from the precursor lactosylceramide via the glycosyl transfer of α1,4-galactosyltransferase (α1,4GT) (JP-A-10-295371). The inventors previously produced a transgenic tobacco generating lactosylceramide via the introduction of human β1,4-galactosyltransferase (β1,4GT5). Because the plant lacks the α1,4 GT gene, the plant cannot produce ceramide trihexoside. This is discussed in co-pending International Patent Application number PCT/JP2005/015899 (Publication No. WO 2006/025443).
A research example is reported, where only one hβ-1,4-GalT 1 (accession No. X55415 or X13223) as an isozyme of β1,4GT is introduced in a liquid culture cell of tobacco, to transfer galactose into the protein (Nirianne Q. Palacpac et al., Stable expression of human β1,4-galactosyl transferase in plant cells modifies N-linked glycosylation patterns, Proc. Natl. Acad. Sci. USA, 96, 4692-4697 (1999)). However, no example about glycosyl transfer into lipid is shown.